Hairpin transcription does not necessarily lead to efficient triggering of the RNAi pathway
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ORIGINAL PAPER
Hairpin transcription does not necessarily lead to efficient triggering of the RNAi pathway Athanasios Dalakouras • Maria Tzanopoulou Mina Tsagris • Michael Wassenegger • Kriton Kalantidis
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Received: 3 February 2010 / Accepted: 4 June 2010 / Published online: 26 June 2010 Ó Springer Science+Business Media B.V. 2010
Abstract Previously, we had shown that stable expression of a hairpin RNA sharing homology with the coat protein (CP) of the Cucumber mosaic virus (CMV) (hpRNACMV) produced CMV resistant Nicotiana tabacum plants. However, only 17% of the hpRNACMV-expressing plants generated substantial amounts of siRNAs that mediated CMV resistance (siRNAsCMV). Here, we demonstrate that the transcription of a hpRNACMV per se is not sufficient to trigger cytoplasmic and nuclear RNAi. A multipletransgene copy line showed a strong resistance
Electronic supplementary material The online version of this article (doi:10.1007/s11248-010-9416-3) contains supplementary material, which is available to authorized users.
phenotype. Segregation of individual copies revealed that in one locus, the transgene-produced hpRNACMV transcript was processed into 21-nt and 24-nt siRNAsCMV and lines containing this locus were resistant. At a second locus, where the transgene was shown to be transcribed, no siRNAsCMV were produced and lines harbouring only this locus were susceptible. In addition, the second locus failed to trigger de novo RNAdirected DNA methylation (RdDM) in cis, of its cognate sequence. However, after being induced in trans, methylation in the transcribed region of the transgene was maintained in both CG and CHG residues. Sequence-specific maintenance of methylation in transcribed regions, as well as diverse RNA degradation pathways in plants are discussed in view of our observations.
A. Dalakouras M. Wassenegger RLP AgroScience GmbH, AlPlanta-Institute for Plant Research, 67435, Neustadt, Germany
Keywords Methylation T-DNA CMV Transgenic Nicotiana benthamiana RNA silencing
A. Dalakouras M. Wassenegger Heidelberg Institute for Plant Sciences, University of Heidelberg, 69120, Heidelberg, Germany
Introduction
A. Dalakouras M. Tzanopoulou M. Tsagris K. Kalantidis (&) Department of Biology, University of Crete, 71409 Heraklion, Greece e-mail: [email protected] A. Dalakouras M. Tzanopoulou K. Kalantidis Institute of Molecular Biology and Biotechnology, Foundation for Research and Technology, 71110 Heraklion, Greece
In plant cells, the presence of a double stranded (ds) RNA molecule can trigger two spatially distinct but functionally interrelated RNA interference (RNAi) responses (Baulcombe 2005; Vaucheret 2006; Voinnet 2008). The Dicer-like (DCL) proteins DCL2, DCL3 and DCL4 process dsRNA into 22-, 24-, or 21-nucleotides (nt) long small interfering RNAs (siRNAs), respectively (Fusaro et al. 2006). In the cytoplasm, 21-nt siRNAs are
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loaded onto an Argonaute1- (AGO1-) containing RNAinduced silencing complex (RISC) which mediates target RNA cleavage and/or inhibition of
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